Abstract
Recent clinical investigations indicate that anthracycline-based chemotherapies induce early decline in heart mass in cancer patients. Heart mass decline may be caused by a decrease in cardiac cell number because of increased cell death or by a reduction in cell size because of atrophy. We previously reported that an anthracycline, doxorubicin (DOX), induces apoptotic death of cardiomyocytes by activating cyclin-dependent kinase 2 (CDK2). However, the signaling pathway downstream of CDK2 remains to be characterized, and it is also unclear whether the same pathway mediates cardiac atrophy. Here we demonstrate that DOX exposure induces CDK2-dependent phosphorylation of the transcription factor forkhead box O1 (FOXO1) at Ser-249, leading to transcription of its proapoptotic target gene, Bcl-2-interacting mediator of cell death (Bim). In cultured cardiomyocytes, treatment with the FOXO1 inhibitor AS1842856 or transfection with FOXO1-specific siRNAs protected against DOX-induced apoptosis and mitochondrial damage. Oral administration of AS1842856 in mice abrogated apoptosis and prevented DOX-induced cardiac dysfunction. Intriguingly, pharmacological FOXO1 inhibition also attenuated DOX-induced cardiac atrophy, likely because of repression of muscle RING finger 1 (MuRF1), a proatrophic FOXO1 target gene. In conclusion, DOX exposure induces CDK2-dependent FOXO1 activation, resulting in cardiomyocyte apoptosis and atrophy. Our results identify FOXO1 as a promising drug target for managing DOX-induced cardiotoxicity. We propose that FOXO1 inhibitors may have potential as cardioprotective therapeutic agents during cancer chemotherapy.
Highlights
Recent clinical investigations indicate that anthracyclinebased chemotherapies induce early decline in heart mass in cancer patients
To determine whether administration of DOX results in forkhead box O1 (FOXO1) phosphorylation in the heart, adult male C57BL/6 mice received a single injection of DOX (5 mg/kg, i.p.) and heart samples were collected at 24 h, a time point showing elevated cardiac cyclin-dependent kinase 2 (CDK2) activity (8)
We demonstrated that DOX exposure induced CDK2-dependent FOXO1 activation, which was necessary for apoptosis and atrophy (Fig. 8C)
Summary
DOX exposure induced FOXO1 phosphorylation at Ser-249 in the heart and cardiomyocytes. In response to DOX treatment, cultured adult mouse cardiomyocytes (AMCMs) exhibited a dramatic increase in phospho-FOXO1 (Ser-249) signal intensity (Fig. 1B), suggesting that DOX exposure induced FOXO1 phosphorylation through a cardiomyocyte-autonomous mechanism. Inhibition of FOXO1 with AS1842856 suppressed DOX-induced cleavage of PARP and caspase-3 (Fig. 4C) and TUNEL labeling (Fig. 4D) These data suggested that FOXO1 activation was necessary for DOX-induced apoptotic death of cardiomyocytes. Oral administration of AS1842856 significantly reduced the protein levels of phospho-FOXO1 (Ser-249) and Bim in the mouse heart, indicating effective FOXO1 inhibition. The number of TUNEL-positive cardiomyocytes in DOX-challenged hearts was significantly reduced by AS1842856 treatment (Fig. 6G), suggesting that inhibition of FOXO1 protected against DOX-induced cardiomyocyte apoptosis. Treatment with AS1842856 abrogated DOX-induced MuRF1 expression, suggesting that FOXO1-mediated transcription of MuRF1 likely mediated DOX-induced cardiac atrophy
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